The innate immune system is critical for host defense but, when dysregulated, can cause severe inflammatory disease. Inflammatory sequelae are mitigated at a number of levels. Principal among these are the precise identification of the threat and the appropriate tailoring of the response. Infectious agents are identified by a variety of pattern recognition receptors that recognize conserved molecular patterns characteristic of the microbe which are not found in the host. Modulation of the response requires multiple levels of regulation that include crosstalk and feedback among various signaling pathways and gene regulatory networks that operate at distinct temporal scales. Systems biology provides a framework in which this complexity can be addressed. In this proposal, we will use systems level analysis to contextualize and elucidate the function of ENU-induced mutations in critical regulatory nodes that impact immune phenotypes. By applying unbiased analytical techniques to global datasets generated from a variety of innate immune cell types under a wide range of conditions, we have identified many genes that are likely to be central regulators of the immune response. While many of these genes are well studied, a significant subset is poorly characterized. Until now, investigation of this latter group of genes has been hampered by the inability to perturb their function and by a lack of knowledge about their interaction with well-characterized pathways. In this proposal we have combined recent advances in ENU mutagenesis methodologies and systems biology in order to bring these genes within reach of mechanistic studies. As ENU-mutated mice are sequenced in the Genetics Core, selected strains carrying mutations in genes predicted by Systems Biology approaches to affect innate immune pathways will be bred to homozygosity in parallel with expansion of the pedigree for large-scale phenotypic screening. Innate immune cells from these mice will be comphrehensively screened for their response to a wide range of stimuli. The function of mutations exhibiting a phenotype is these experiments will then be examined in detail using a broad range of systems biology approaches.